I. Field of the Invention
The present invention relates to the fields of molecular and cell biology generally, and more specifically, it addresses mechanisms for growth control in eurkaryotic cells. In particular, there are provided viral genes that inhibit normal cell death and methods for use thereof.
II. Related Art
The control of host cell gene expression, and often the control of genes involved in DNA replication, are integral parts of the life cycle of a virus. However, recent evidence suggests that most eukaryotic cells respond to viral disruption of normal cellular physiology by undergoing programmed cell death (apoptosis) (White, 1993). To counteract this, many viruses have evolved mechanisms to block host cell death (Clem and Miller, 1994; White and Gooding, 1994). In several cases, viral genomes have been found to contain genes whose products interact with proteins that play a central role in regulating cell survival.
Programmed cell death is triggered by several factors and may take various forms. For example, the synthesis of double-stranded RNA activates kinases which phosphorylate the xcex1 subunit of eIF-2 and completely turn off protein synthesis (Sarrel, 1989). Ultimately, activation of metabolic pathways causes a pattern of morphological, biochemical, and molecular changes which result in cell death without spillage of cellular constituents which would result in an inflammatory response detrimental to the host (Wyllie, et al.).
Apoptotic cell death is commonly observed during embryogenesis and organ involution and in the natural death of terminally differentiated cells at the end of their life span. Most viruses which induce either the shut-off of protein synthesis or apoptosis also have evolved mechanisms which block host responses and enable them to replicate in their hosts (Shen and Shenk, 1995). Among the best-known examples of viral gene products which block apoptosis are the adenovirus E1B Mr19,000 protein (Rao, et al, 1992.), vaccinia CrmA protein (Ray, et al.), simian virus 40 (SV40) T antigen (McCarthy, et al., 1994), human papillomavirus No. 16 (HPV 16) E6 protein (Pan and Griep, 1994), Epstein-Barr virus BHRF1 protein (Henderson, et al., 1993) and human cytomegalovirus IE1 and IE2 gene products (Zhu, et al., 1995). Herpes simplex virus 1 (HSV-1) encodes a protein, xcex3134.5, which blocks the phosphorylation of eIF-2xcex1 (Chou and Roizman, 1992).
The utility of proteins that are capable of inhibiting apoptosis are manifold. First, such proteins, or their corresponding genes, may be used to immortalize cell lines that otherwise would perish during culture. This makes possible not only the study of these cells, but also presents the option of growing these cells in large numbers in order to isolate protein species therefrom. Second, the identification of inhibitors of apoptosis and their function permits the possible intervention, in a clinical setting, when these proteins are interfering with normal programmed cell death, or apoptosis. This may be accomplished by providing an inhibitor or an antisense nucleic acid that interferes with the expression of a protein that interferes with apoptosis. Thus, the identification of novel proteins having these activities and uses provide important new tools for those working in this arena.
It is, therefore, an object of the present invention to provide methods for the use of infected cell protein number 4, or ICP4, and its cognate gene xcex14, as inhibitors of apoptosis. In addition, it is an object of the present invention to provide methods for the use of agents that inhibit ICP4 and/or xcex14 in order to induce apoptosis in HSV infected cells. It also is an object of the present invention to provide methods for the identification of agents that inhibit the apoptosis-inhibiting function of ICP4.
In satisfying these goals, there is provided a method for blocking apoptosis of a cell comprising the step of providing to the cell an HSV ICP4 polypeptide or an HSV xcex14 gene. The xcex14 gene may be contained in an expression vector and, further, under the control of a promoter active in eukaryotic cells. One such promoter is the a tetracycline controlled promoter. The expression vector further comprises a selectable marker and/or further comprises a gene encoding a second polypeptide under the transcriptional control of a promoter active in eukaryotic cells.
In another embodiment, there is provided a method for inducing apoptosis in a cell infected with HSV comprising the step of administering to said cell an agent that inhibits HSV ICP4 function in said cell. The agent may inhibit transcription or translation of an HSV xcex14 gene or transcript or may bind to an HSV ICP4 polypeptide. The reagent may be an antisense HSV xcex14 construct an antibody that binds immunologically to an HSV ICP4 polypeptide. Particular antisense constructs are oligonucleotides that hybridizes to a 5xe2x80x2-untranslated region for an HSV xcex14 gene or a translational start site for an HSV xcex14 transcript. Particular antibodies are polyclonal sera against ICP4 or a monoclonal antibody against ICP4.
In yet another embodiment, there is provided a method for treating a subject with an HSV infection comprising the step of inhibiting HSV ICP4 function. The inhibition may comprise providing to the subject a first pharmaceutical composition comprising an HSV xcex14 antisense construct or a monoclonal antibody that binds immunologically to an HSV ICP4 polypeptide. The first pharmaceutical composition may be applied topically to HSV infected cells in said patient. The method may further comprise the step of providing to said subject a second pharmaceutical composition comprising a conventional anti-HSV agent, such as acyclovir. Acyclovir is delivered via a route selected from the group consisting of topically, orally and intravenously.
In yet still another embodiment, there is provided a screening method for compounds having inhibitory activity against HSV ICP4 polypeptide-induced inhibition of apoptosis comprising the steps of (a) providing a first cell comprising an HSV xcex14 gene under the control of an HSV immediate early promoter; (b) infecting said first cell with a herpes simplex virus that lacks a functional xcex14 gene; (c) contacting said first cell with a test compound; (d) incubating said first cell under conditions permitting viral replication; and (e) comparing the cell pathology of said first cell following incubation with the cell pathology of a second cell that lacks an HSV xcex14 gene following infection with said herpes simplex virus and the cell pathology of a third cell comprising an HSV xcex14 gene under the control of an HSV immediate early promoter following infection with said herpes simplex virus but in the absence of said test compound. Cell pathology comprises condensation of chromatin, obliteration of nuclear membranes, vacuolization, cytoplasmic blebbing and DNA fragmentation.
The screening method may employ a cell line which contains an integrated copy of a wild-type HSV xcex14 gene under the control of an xcex14 promoter and a herpes simplex virus that lacks a functional HSV xcex14 gene has a deletion in both copies of the virally-encoded xcex14 genes. For example, the herpes virus may carry a temperature sensitive mutation in both copies of the virally-encoded xcex14 gene; incubation is at 39.5xc2x0 C.
Another screening method for compounds having inhibitory activity against HSV ICP4-induced inhibition of apoptosis comprises the steps of (a) providing a first cell comprising an HSV xcex14 gene under the control of an inducible promoter; (b) inducing transcription from said promoter; (c) contacting said first cell with a test compound; (d) incubating said first cell under conditions expression of an HSV ICP4 polypeptide; and (e) comparing the cell pathology of said first cell following incubation with the cell pathology of a second cell not having an HSV xcex14 gene following induction and the cell pathology of a third cell comprising an HSV xcex14 gene under the control of an inducible promoter following induction but in the absence of said test compound.
Yet another screening method for compounds having inhibitory activity against HSV ICP4 polypeptide-induced inhibition of apoptosis comprises the steps of (a) providing a first cell; (b) infecting said first cell with a herpes simplex virus; (c) contacting said first cell with a test compound; (d) incubating said first cell under conditions permitting viral replication; and (e) comparing the cell pathology of said first cell following incubation with the cell pathology of a second cell treated with said test compound alone and the cell pathology of a third cell following infection with said herpes simplex virus but in the absence of said test compound.
In still yet a further embodiment, there is provided a method for expressing a polypeptide in a cell comprising the steps of (a) contacting a cell with a herpes virus vector encoding said polypeptide; (b) contacting said cell with an agent that blocks the transactivating function of ICP4 but does not block the apoptosis inhibiting function of ICP4.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.